Three-dimensional microtissues with integrated mechanical loading

ABSTRACT

This document describes systems and methods for integrated mechanical loading of tissue. The system includes a three-dimensional tissue comprising organic material. The system includes a strip of bendable material. The strip includes a first region proximate to a first end of the strip coupled to the tissue. The strip includes a second region near a second end of the strip for coupled to the tissue, the second end being opposite the first end, wherein the tissue exerts a force on the strip to bend the strip, the force caused by contraction of the tissue, and wherein the strip exerts a stress on the tissue.

CLAIM OF PRIORITY

This application claims priority under 35 U.S.C. § 119(e) to U.S. PatentApplication Ser. No. 62/605,475, filed on Aug. 15, 2017, the entirecontents of which are hereby incorporated by reference.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with government support under HL117750 awardedby National Institute of Health. The government has certain rights inthe invention.

TECHNICAL FIELD

This document relates to tissue generation.

BACKGROUND

Engineered three-dimensional (3D) in vitro models of functional humantissues have applications as physiologically relevant but moreeconomical platforms for pharmacological testing (compared to human andanimal trials), disease models and disease modeling (e.g., geneticallytailored disease modeling), and building blocks to engineer functionalorgan replacements in the future. Animal models do not typicallyaccurately predict the response pharmacological stimuli will have onhuman patients. 2D in vitro cultures of human cells do not accuratelypredict the response pharmacological stimuli will have on humanpatients. For example, a process for engineering in vitro 3D cardiacmuscle from human stem cell derived cardiomyocytes (CMs) instead ofanimal CMs enables engineered cardiac muscle that is physiologicallymore similar to adult human heart muscle. Further, engineered cardiacheart muscle that is representative of human physiology would fill acritical void between animal models and human clinical trials. However,human adult CMs are terminally differentiated and cannot be expanded invitro. The only robust source of new human CMs are from pluripotent stemcells including either embryonic stem cells (ESCs) or inducedpluripotent stem cells (iPSCs). In fact, much in vitro understanding ofcardiac function has focused on individual CM behavior. Specifically,focus has been placed on differentiating more functionally mature CMsfrom ESCs or iPSCs. Additional studies have examined individualpluripotent stem cell-derived CM response to different pharmacologicalstimuli in terms of electrical activity, force generation, and geneexpression. The challenge with iPSC and ESC-derived CMs is that they areoften more functionally immature than in vivo adult CMs. Specifically,these CMs are often round rather than spread and classically rod-shaped,exhibit disordered sarcomere organization, and have smaller membranecapacitance than adult CMs. Determining how to differentiate morefunctionally mature CMs from stem cells is key to engineering functionalcardiac tissues, but a necessary next step is developing more complex,physiologically representative, 3D microtissues (tissues) by building onthese individual CM studies.

Furthermore, current preclinical research for treatment and diseasemodeling is often performed on animals or 2D cell cultures. MuscularThin Films (MTFs) and cardiac tissues are exposed to a static load thatmay not accurately model diseases in which load on the heart muscleincreases and are isometrically constrained.

SUMMARY

This document describes methods and systems for generating 3D tissuesthat are integrated with mechanical loading. The tissues generated arerepresentative of human physiology and we describe and example thatincludes differentiated, functionally mature muscle tissues such as CMsexhibiting striations. Tissues are fabricated with an attached strip ofmaterial (e.g., a PDMS strip), which can be used to load the tissue andmeasure tissue contractile force. The material strip has knownmechanical properties, and the strip bending is quantified to determinetissue contractile force of the generated tissue. For example, stripbending is measured by taking one or more images of the strip andtissue, processing the images to determine the geometry of the stripwith respect to the tissue, and determining the strip contractile forcebased on the determined geometry of the strip. The tissues are referredto as mircrotissues (μtissues) and/or 3D tissues because the tissueshave a length, width and thickness and are not grown as two-dimensionalcell cultures on cover slips or films. The tissue can include muscletissues (cardio, skeletal, etc.), tendons, ligaments, cartilage, skinand so forth.

In some implementations, strip is bent in to a semi-circle shape and thetissue is formed across the ends, in a horse shoe like configuration.The tissue contractile force is determined by measuring the radius ofcurvature of the strip and its length with respect to the tissue.Measurements in tissue length and force are plotted over time. A twitchforce is determined based on the difference between an average systolicforce and an average diastolic force of the tissue. Thus, because thestrip can be viewed with a standard microscope, this process includes anon-invasive method of determining tissue contractile force of thegenerated tissue, in comparison to the laborious process of connectingthe microtissue to a mechanical force probe.

The advantages of the tissues and methods for generating herein aredescribed below. Cells grown in vitro in 2D are by definition adhered toa cover slip or other substrate. This adhesion to a substrate limits theability of the cells to recapitulate the structure and function of real3D tissues in the human body. Higher cell-substrate interactions (suchas those in 2D and similar deformable 2D systems such as muscular thinfilms) are unsuitable for modeling cardiac disease phenotypes drivenpredominantly by defects in cell-cell or cell-matrix interactions. While2D cells are isometrically constrained because they are adhered to rigidsubstrate that they cannot deform. Rather, the 3D tissues describedbelow are not constrained by an underlying substrate and therefore freeto contract and shorten in length in response to a stimuli, producing acontractile stress that works against the mechanical loading of thestrip. Tissues are fabricated with an attached strip of bendablematerial (e.g., PDMS material, a thermoplastic such as Teflon film,polycarbonate film, etc.), whereby strip bending is quantified todetermine tissue contractile force. The parameters of the strips, whichinclude length, elastic modulus, width, thickness, and so forth, aretuned to particular values to create a defined mechanical load. Thisparameter space is useful for generating many variations of the tissueand the mechanical loading for high-throughput assay (HTA) processes.Specifically, the parameters are altered to change the bending stiffnessof the strip, making each well of an assay plate unique, if needed. Suchapplications include modeling cardiac diseases, which often are causedby of produce an altered loading of the heart. In a specific example, aPDMS strip has tunable bending stiffness, in which the PDMS stripparameters are changed to alter the bending stiffness that the cardiactissue must contract against. This enables modulation of the load acardiac tissue experiences. The load can be tuned to be on the order of˜1 kPa to 10,000 kPa.

Because the 3D tissue attached to the strip is not isometricallyconstrained, this allows the tissue to undergo large strains of about10-20%, which is more reminiscent of the adult myocardium. As thecardiac tissue contracts it bends the PDMS strip. As stated above, thecurvature of the strip is measured in a noninvasive manner (e.g., bybrightfield microscopy and image processing) to quantity tissuecontractile force without disturbing the tissue contraction. It is alsopossible to modulate between constrained and unconstrained condition,which enables tissue development where the tissue can contract in excessof 40% of an initial length of the tissue. This tissue thus closelyresembles adult myocardium (or other such tissue that is generated) thatcan be interrogated to acquire data such as twitch rate, contraction,structure, histology, force generation, electrophysiology, conductionvelocity, gene expression, protein expression, or other data acquiredfrom such assays performed on tissues.

The system includes a strip of bendable material, the strip including: afirst region in proximity to a first end of the strip for coupling to atissue including organic material; and a second region in proximity to asecond end of the strip for coupling to the tissue, the second end beingopposite the first end, where the strip is configured to bend to alignthe first region with the second region; and a well for generating thetissue, the well including: a region for generating the tissue from acell culture; a first slit configured to receive the first end of thestrip and expose the first region of the strip to the tissue generationregion; and a second slit configured to receive the second end of thestrip and expose the second region of the strip to the region of thewell; where the first slit is aligned with the second slit to align thefirst region of the strip and the second region of the strip in theregion to enable the tissue to couple to the first region of the stripand to couple to the second region of the strip during generation of thetissue.

In some implementations, the well is configured to reduce a stressexerted by the strip from on the tissue during generation of the tissuerelative to a maximum stress that the strip is configured to exert onthe tissue; and where the strip is configured to exert the maximumstress on the tissue when the strip and the tissue are removed from thewell. In some implementations, the strip is configured to provide astress of up to approximately 10,000 kPa on the tissue when the stripand the tissue are removed from the well.

In some implementations, a magnitude of the stress exerted on the tissueby the strip is a function of one or more tuned parameters of the strip,the parameters including a length of the strip, a width of the strip, athickness of the strip, an elastic modulus of the strip, and a shape ofthe strip. In some implementations, the tissue is configured forcontracting between approximately 10%-40% of an initial length of thetissue.

In some implementations, a cell suspension of the well comprises anapproximate mixture of either 0.5 to 10 mg/mL Collagen Type I or fibrin,20% Matrigel, 10% 10× phosphate buffered saline, and either 18.75×10⁶cells/mL for cardiomyocytes or 15×10⁶ cells/mL for myoblasts. A cellsuspension of the well comprises a concentration of betweenapproximately 10-100×10⁶ cells/mL and fibroblasts includingapproximately 10-20% of a total cell count. In some implementations,cells of the cell suspension comprise one of smooth muscle cells, skincells, ligament cells, and tendon cells.

In some implementations, the well is a part of a multi-well plate. Insome implementations, at least one well of the multi-well platecorresponds to a respective strip having particular parameters, andwhere at least one strip and well of the multi-well plate represent aloading value of a parameter space representing loading values for thetissue.

In some implementations, the particular parameters of the strip comprisean elastic modulus parameter, a thickness parameter, a width parameter,and a length parameter. In some implementations, the strip comprises oneof polydimethylsiloxane (PDMS), Teflon film, or a polycarbonate film. Insome implementations, each of the first region and the second region ofthe strip has a narrower width than a width of a portion of the stripbetween the first region and the second region.

In some implementations, the tissue comprises one of a cardiac tissue,skeletal tissue, smooth muscle tissue, skin tissue, cartilage, tendon,and ligament. In some implementations, the tissue forms striations inresponse to a stress exerted on the tissue by the strip. In someimplementations, cells within the tissue align in response to a stressexerted on the tissue by the strip. The tissue is configured to undergoa strain of up to 80% by the strip when the strip and tissue are removedfrom the well.

In some implementations, a process for generating a tissue with anintegrated load includes generating a tissue that is affixed to a stripof bendable material, the tissue being affixed to a first end and asecond, opposite end of the strip. The process includes causing thetissue be in a contracted state and exert a stress on the strip to bendthe strip. The process includes measuring a curvature of the strip whenthe tissue is in the contracted state and exerting the stress on thestrip. The process includes calculating the stress exerted on the stripby the tissue, the stress being a function of the curvature of the stripand one or more parameters of the strip, the one or more parameters eachhaving a value that is pre-determined.

In some implementations, the process includes tuning an action potentialof the tissue by adjusting the one or more parameters of the strip;applying a voltage to the tissue; and responsive to application of thevoltage, measuring the action potential of the tissue using calcium orvoltage imaging. In some implementations, the process includes measuringan organization of cell cytoskeletal components. In someimplementations, the process includes measuring an epigenetic change inthe tissue. In some implementations, the process includes measuring agene or protein expression of the tissue. In some implementations, theprocess includes measuring a gene or protein expression of the tissue.

In some implementations, the process includes controlling, during thegenerating of the tissue, a density of the tissue by adjusting aconcentration of a cell culture in a hydrogel mixture. In someimplementations, the hydrogel mixture includes at least one offibrinogen, Matrigel, a hyaluronic acid hydrogel, or a synthetichydrogel.

In some implementations, at least one of the one or more parameterscomprise a thickness of the strip, a width of the strip, an elasticmodulus of the strip and a length of the strip. The process furtherincludes selecting the value of the one or more parameters to tune amagnitude of a stress exerted on the tissue by the strip to a particularvalue.

In some implementations, the process includes adding a compound to thetissue so that the tissue absorbs the compound and causing the tissue bein a contracted state and exert a stress on the strip to bend the striponce the compound is absorbed by the tissue. The compound comprises adrug candidate. In some implementations, the process includes adding acompound to the tissue so that the tissue absorbs the compound; andcausing the tissue be in a relaxed state once the compound is absorbedby the tissue so that the strip extends the tissue.

In some implementations, a process for generating a tissue includesadding, to a well, a cell suspension mixture, the well including a stripof bendable material, where the strip of bendable material is insertedinto the well at a first end and at a second end opposite the first endso that the strip is curved; generating, from the cell suspensionmixture, a tissue that is affixed to the first end of the strip and thesecond end of the strip; and removing the strip from the well, where thestrip is configured to exert a stress on the tissue after the strip isremoved from the well, and where the strip exerts a reduced stress onthe tissue before the strip is removed from the well relative to anincreased stress on the tissue after the strip is removed from the well.

In some implementations, the system includes a three dimensional tissueincluding organic material; and a strip of bendable material, the stripincluding: a first region proximate to a first end of the strip coupledto the tissue; a second region near a second end of the strip forcoupled to the tissue, the second end being opposite the first end,where the tissue exerts a force on the strip to bend the strip, theforce caused by contraction of the tissue, and where the strip exerts astress on the tissue.

In some implementations, the tissue is isometrically unconstrained. Insome implementations, the tissue is configured to contract by at least10% an initial length of the tissue. In some implementations, the tissueis configured to contract by at least 20% an initial length of thetissue.

In some implementations, a process includes selecting one or moreparameters of a strip to tune a loading value of the strip that thestrip is configured to exert; generating a tissue that is integratedwith the strip that provides the loading value on the tissue; adding acompound to the tissue so that the tissue absorbs the compound; andmeasuring an effect of the compound on the tissue.

In some implementations, the compound is a drug including a muscarinicagonist. In some implementations, the compound is a drug including astimulant. In some implementations, the process includes measuring theeffect comprises measuring one or more of an organization of cellcytoskeletal components of the tissue, an epigenetic change in thetissue, and a gene or protein expression of the tissue.

In some implementations, the one or more parameters comprise a length ofthe strip, a width of the strip, a thickness of the strip, a shape ofthe strip, and an elastic modulus of the strip. In some implementations,the tissue is one of a plurality of tissues, and where one or more theplurality of tissues each experience a different loading value. In someimplementations, one or more of the plurality of tissues are combinedwith different compounds

The details of one or more embodiments of the microtissues are set forthin the accompanying drawings and the description below. Other features,objects, and advantages of the microtissues and methods for generatingthem will be apparent from the description and drawings, and from theclaims.

DESCRIPTION OF DRAWINGS

FIG. 1A shows a top view of a well and a top view of a strip of bendablematerial.

FIG. 1B shows a system for generating tissue.

FIGS. 2A-2B show examples of tissue generation.

FIG. 3 shows a perspective view of the tissue generation of FIGS. 2A-2B.

FIGS. 4-5 show examples of wells, well plates, and strips.

FIG. 6 shows images of cardiac muscle tissues with cardiomyocytes andwith 0%, 10% and 20% cardiac fibroblast cocultures.

FIGS. 7A-7B show images of immunofluorescence staining of cardiacmicrotissues.

FIG. 8 shows tissue structure and cell organization.

FIG. 9 shows finite element modeling of a strip.

FIG. 10 shows an example well plate.

FIG. 11 shows a process for determining the contractile force of atissue in a non-invasive manner.

FIG. 12 shows a process for generating the tissue.

FIG. 13 shows a contractility assay system for determining thecontractile force of a tissue in a non-invasive manner.

FIG. 14 shows twitch forces and beat frequencies of a generated tissue.

FIGS. 15-16 shows twitch forces and beat frequencies of a tissue withvarious chemical stimuli introduced.

FIG. 17 shows measured cross-sectional areas of tissue samples.

FIG. 18 shows measured twitch forces normalized for tissuecross-sectional area.

FIG. 19 shows stress generation of a tissue relative to concentration ofcollagen used to form the 3D tissue and to the thickness of the stripused to provide a mechanical loading to the tissues.

FIG. 20 shows images for determining stress generation of a tissue in anon-invasive manner where the tissue length, length of the strip betweentissue attachment points and curvature of the strip are identified usingan image analysis program.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

Systems and methods for generating 3D microtissues with integratedmechanical loading are described in reference to FIGS. 1A-2B, below.

FIG. 1A shows a top view of a well 100 for generating 3D tissues withintegrated loading. FIG. 1A also shows a top view of a strip 112 ofbendable material. The well 100 includes a well wall 102, a tissuegeneration region 104 for generating the tissue from a cell culture. Thewell 100 includes a first slit 106 a that receives a strip 122 at afirst end 120 a of the strip. The well includes a second slit 106 b thatreceives a second end 120 b of the strip 112. The first slit 106 a andthe second slit 106 b are approximately aligned in the well 100. Thefirst and second slits 106 a-b are spaced in the well at a length 108.The length 108 is based on a size of the well 100. The length 108determines what an initial tissue length will be of tissue generated inthe well 100. The tissue being generated includes an initial length(e.g., before contraction of the tissue) approximately equal to length108. Length 108 is typically less than 60 mm (e.g., 10 mm, 20 mm, or andsuch length required for generating tissue of a desired length), but canbe expanded beyond this size for large wells.

A strip 112 of bendable material is configured to be inserted into slits106 a, 106 b of the well. The strip is typically formed of PDMSmaterial, but other materials can be used. Here, the term “bendable”refers to any material that is capable of bending and exerting a stressforce in compression on a tissue attached to either end of the strip112. For example, the strip 112 can include a medical grade elastomer.For example, the strip 112 can include a thin thermoplastic above theT_(g) such as Teflon film, polycarbonate film, elastomeric film, etc.When bent, the strip 112 is configured to provide a load based on theparameters (e.g., length, width, thickness, shape, elastic modulus,etc.) of the strip. The load (e.g., stress on the tissue) provided bythe strip 112 can range from ˜1 kPa to ˜10,000 kPa. The load can betuned based on selection of the parameters of the strip 112. In this waya particular load on the tissue can be selected to provide a stressforce on the tissue of a particular value.

The strip 112 can include any geometry needed to tune the strip to exerta particular load on the tissue with which it is integrated. Thegeometry of the strip 112, including the length 114, width 116, andthickness (dimension 122 of FIG. 1B), are each variable parameters thataffect the load that the strip 112 exerts on an integrated tissue. Afirst end 120 a of the strip 112 is inserted in slit 106 a of well 100,and a second end 120 b is inserted in slit 106 b of well 100. The strip112 is configured to bend when inserted into well 100. The length 114 isselected for determining how much the strip 112 bends when inserted intothe well 100. Each end 120 a, 120 b is sized to fit precisely into slits106 a, 106 b, respectively, so that no cell culture mixture enters theslits.

The well 100 includes a tissue generation region 104 where a mixture ofcells with a hydrogel for culture is disposed in the well. The strip 112includes a first region 118 a proximate the first end 120 a of the stripfor coupling to the tissue of organic material. The strip 112 includes asecond region 118 b proximate the second end 120 b of the strip forcoupling to the tissue. The first and second regions 118 a, 118 b areexposed to the cell and hydrogel mixture when the strip 112 is insertedinto the well 100. In some implementations, the first and second regions118 a,118 b are narrower, necking regions compared to the width 116 ofthe strip 112. The narrower width facilitates tissue attachment to thestrip once the tissue forms from the cell culture.

FIG. 1B shows a side view of a system 130 for generating tissueincluding the well 100 of FIG. 1A and the strip 112 of FIG. 1A. Thestrip 112 is bent and inserted into slits 106 a, 106 b of the well 100.The tissue generation region 104 is shown as only partially filling thewell 100. However, the well 100 can be fully filled with a cell culture,and a greater portion of the strip 112 can be exposed to the cellculture. The slits have a depth denoted by dimension 126. Typically, theslits are less than 10 mm deep, but can be made larger for longerstrips. Strip thickness 122 can vary and determines how much load thestrip 112 exerts on the tissue once the strip and tissue are removedfrom the well 100 (e.g., in addition to strip length, width, elasticmodulus, and shape). While inserted into the well 100, the slits 106a,106 b prevent the strip 112 from moving and limits the load of thestrip 112 from being exerted on the tissue. The tissue contracts againstthe strip while the strip is inserted in the well, and the first andsecond regions 118 a, 118 b arc approximately fixed in place. When thestrip 112 and tissue are removed, the full loading of the strip isexerted on the tissue.

FIGS. 2A-2B show examples of tissue generation. In FIG. 2A, the strip112 has been removed from the well 100 and the tissue 200 iscontracting, pulling the ends of the strip 112 together to a length 204that is shorter than length 108 of FIGS. 1A-1B. In some implementations,the tissue 200 contracts by at least 10% of an initial length 108 (e.g.,so that length 204 is less than 90% of length 108, respectively). Insome implementations, the tissue 200 contracts even more, shortening atleast 40% of an initial length 108 (e.g., so that length 204 is 60% oflength 108, respectively). Arrows 206 show a stress load exerted on thetissue 200 by the strip 112. Arrows 208 show a contractile force of thetissue. The contractile force 208 can be measured by determining howmuch the strip 112 has bent during contraction of the tissue 200. Thismeasurement, in combination with the selected dimensions of the strip112, form a basis for calculating the contractile force 208 of thetissue in a non-invasive manner. For example, no strain gauges need beinserted into the tissue—the measurement process can be passive. Thecontraction shown in FIG. 2A can be the result of applying electricalstimulation to the tissue 200, as described in further detail, below.

In some implementations, the tissue 200 contracts to a length 204 thatcan be approximately 10% shorter than an initial length of the tissue.In some implementations, the tissue 200 contracts even more (up to 40%an initial length of the tissue). The tissue is affixed to the strip 112at regions 202 a and 202 b of the tissue. As stated above, the ends ofthe strip 120 a, 120 b can be wider than the regions of the strip 112 towhich the tissue is affixed to ensure that the tissue does not slip offthe strip once the strip is removed from the well.

FIG. 2B shows a strip 112 integrated with tissue 210. Arrows 206 show astress load of the strip 112 on the tissue 210. Arrows 212 show acontractile force of the tissue 210. Length 210 can be approximately thesame as length 108, or between lengths 108 and 204. Regions 202 a-b ofthe tissue are affixed to the strip 112. The tissue 210 may contractless than tissue 200 of FIG. 2A for several reasons. For example, tissue210 may contract less over time, or a lesser contractile force of tissue210 may be indicative of disease, etc. The longer length 210 between theattachment regions 202 a, 202 b cause the strip 112 to curve less thanin FIG. 2A. The reduction in the curvature can be imaged and thecontractile force 212 can be compared to a nominal contractile force(e.g., force 208).

A particular embodiment of the tissue generation of FIGS. 1A-2B is nowdescribed. A contractile 3D CM tissue is generated as shown in FIG. 3.The potential of the generated tissue as a cardiac model tissue is shownbelow. The tissue functions as a template to engineer 3D skeletal musclemodels. The tissue generation system is configured to enable measurementof twitch forces of the contractile tissues (e.g., also described asμtissues or microtissues), without invasive probes or force transducers.Specifically, a thin, U-shaped, PDMS strip is integrated with 3D tissuesand used the change in bending or tissue length as a means to measuretwitch force. Images 300 include views of the PDMS strip (e.g., strip112), PDMS well base (e.g., well 100 of FIG. 1A), and muscle μtissue(e.g., tissue 200 of FIGS. 2A-2B) as they appear during culture. Thethin, PDMS strip had a narrow necking region (e.g., attachment regions118 a-b of FIG. 1B) tissue attachment. A wide base at each end of thestrip (e.g., ends 120 a-b of FIG. 1B) is hidden in slits in the PDMSwell base to constrain the tissue to attach to the necking region of thestrip.

Tissue contraction induces bending of the PDMS U-shaped strip, and theforce required to induce changes in bending is measured by a simple beamor cantilever bending model, or optionally a more complicated mechanicalmodel of finite element model, of the PDMS strip. Images 310, 320 showthe tissue as removed from the well. The wide base of the strip allowedthe tissue to be removed from the well with the strip intact to performa contractility assay. Upon contraction of the tissue, a force (marked Fin FIG. 3) induces bending of the PDMS strip.

In some implementations, human ESC-derived CMs are used to engineerinitial tissues termed muscle with integrated force indicators (MIFIs).During experimentation, the cardiac MIFIs responded as expected toexcitatory pharmacological stimuli and have the potential to serve as invitro models of human CM function upon further characterization.

The MIFI model serves as a template that can be applied to iPSC-derivedCMs for patient specific disease modeling or skeletal muscle myoblastssince the well and strip design readily translate to other contractilecell types. This system has an advantage over traditional PDMSpost-bending based tissues because the strip design enables easy removalof the MIFIs from culture wells without damaging or altering tissuearchitecture, and also enables the tissue to contract in anon-constrained manner. This allows investigation (e.g., high-throughputassay investigation) of the effect of increased mechanical load on thesecardiac or skeletal muscle model tissues. The strips generate a stressfield that influences cells of the tissue to align along the long axisof the well. Initially, full force of the strip is partially hiddenbecause the PDMS well base does holds the ends of the strip in placeduring culture. When the MIFI tissues are removed from the PDMS wells,the applied load to the MIFI increases, forcing the cells to workagainst the strip to maintain tissue architecture. Thus, this systemserves not only as a tool to better understand cardiac and skeletalmuscle tissue in normal and disease states but to also allowinvestigation of combinatorial effects of various small molecule or drugtherapies with muscle exercise. Alternatively, this system serves fortesting the effects of one or more drug compounds alone, independent ofmuscle exercise.

To generate the tissue, well molds are designed (e.g., well 100 of FIG.1A) for casting PDMS for tissue culture. Initial prototypes can beprinted with acrylonitrile butadiene styrene (ABS) plastic. Final moldscan be printed with a 3D printer.

In some implementations, to create wells from plastic molds, Sylgard 184PDMS (Dow Corning) is mixed at a 10:1 weight ratio of base to curingagent at a 2 minute 2000 RPM mixing cycle and a 2 minute 2000 RPMdefoaming cycle. Universal mold release spray is sprayed onto plasticmolds to facilitate tissue removal. PDMS is poured until the molds arefilled. In some implementations, PDMS wells are degassed for 30 minutesand cured in a 65° C. oven for 2 hours before being removed from theplastic molds.

Well strip (e.g., strip 112) geometries are selected based on the targetstress to induce on the tissue. The strip is cut from a sheet of pliablematerial (e.g., PDMS or other similar material). In someimplementations, dogbone-like strips are formed from non-reinforced,0.005″ thick medical PDMS sheeting, though the parameters of the strip(e.g., length, width, elastic modulus of the material etc.) can beadjusted based on a target stress (in other words, the load that thetissue contracts against). In some implementations, the strips aremounted onto glass microscopy slides and imaged with a VHX-5000 digitalmicroscope to measure thickness. Typically, the average of threemeasurements for each strip is used for later analysis in thecontractility assay.

For verifying elastic modulus of the strips, the strip samples aremounted on a uniaxial tensile testing machine (e.g., using an Instron)and strained at a rate of 1 mm/minute until >10% strain was reached.Sample cross-sections are calculated from average optical measurementsof the individual strips prior to testing. Typically, strip thicknessesare obtained by averaging three random thickness measurements per strip(e.g., using a VHX-5000 digital microscope). Stress is calculated bydividing measured force by measured cross-sectional area. Stress isplotted against strain, and the linear curve fit is obtained for thefirst 10% of strain. For example, slopes of the curves of sample stripswere averaged to obtain a final measurement of 3.10 MPa for the elasticmodulus of PDMS strips. The elastic modulus was used for modeling theforce required to bend strips to specific conformations for thecontractility assay.

A particular implementation for culturing cells for tissue generation isdescribed below, but other similar implementations are possible. Cellsfor cell cultures are typically kept in continuous culture in 6-welltissue culture treated plates pre-coated with 12 μg/cm2 of Geltrex™ andkept at 37° C. and 5% CO₂. In some implementations, all media aresupplemented with 1:1000 Mycozap-CL (Lonza). In some implementations, topre-coat well plates, Geltrex™ is diluted in 4° C. DMEM/F12 to a finalconcentration of 114 μg/mL before pipetting 1 mL/well. Plates are thenincubated with Geltrex™ at room temperature for at least 1 hour priorcell seeding or are stored at 4° C. for up to 1 week. In someimplementations, cells are rinsed with 1×PBS (GE Healthcare) andincubated with TrypLE Express for 5 minutes. The cells are then detachedby pipetting, transferred to DMEM/F 12 stopping media and centrifuged at200 G for 5 minutes. In some implementations, media are aspirated fromthe cell pellet, and cells were re-suspended and seeded at 13.1×10³cells/cm² in Essential 8 media (E8) supplemented with 5 μM Y27632(System Bioscience). After 24 hours Y27632 supplemented E8 was exchangedfor E8 media daily until cells reached 80% confluence, at which pointcells were passaged or differentiated.

For preparing the cell cultures, the following process is typical, butsimilar processes are possible. To induce differentiation, on day 0,cells are rinsed with 10×PBS and 3 mL/well of RPMI 1640 basal mediasupplemented with 1:50 B27 without insulin (RPMI/B27) and 6 μMCHIR99021(LC Laboratories). On day 2, cells are washed with 1×PBS before adding 3mL/well of RPMI/B27 with 2 μM Wnt-059 (Selleck Chemicals). On days 4 and6, the media are exchanged for 3 mL/well of RPMI/B27. On days 8 and 10,the media are exchanged for 3 mL/well of CDM3: RPMI 1640 basal mediasupplemented with 500 μg/mL of O. Saliva derived recombinant humanalbumin (RHA) (Sigma-Aldrich) and 213 μg/mL of L-ascorbic acid2-phosphate (AAP) (Sigma-Aldritch). At day 12, if CMs are visiblybeating, cells were passaged for purification.

For purification, differentiated cells are passaged by washing with1×PBS and incubated in TrypLE Express for 15 minutes at 37° C. Cells arereleased from plates by pipetting into DMEM/F 12 stopping media (2mL/well) and are then centrifuged at 200 G for 7 minutes. Cells areseeded on Matrigel™ (Corning) coated 6 well plates. The plates werecoated with Matrigel™ following the same protocol used to coat plateswith Geltrex™. The cells are resuspended in CDM3L (RPMI 1640 withoutD-glucose supplemented with 500 μg/mL RHA, 213 μg/mL AAP, 7.1 mM sodiumDL-lactate (Sigma-Aldrich) and 5 μM Y27632) media. Three mL/well ofCDM3L (without Y27632) was then exchanged 24 hours after seeding and atday 4. At day 7, cells were passaged by rinsing with 1×PBS, incubatingfor 15 minutes in TrypLE Express at 37° C., centrifuged at 200 G for 7minutes, and were then used for experiments.

Human Cardiac Ventricular fibroblasts are used at <15 doublings, and arecultured at 37° C. and 5% CO². Cells are cultured in FB Growth Medium-3(FGM3) made from the FGM-3 BulletKit™ (Lonza) consisting of FBMsupplemented with 0.1% rhFGF-B, 0.1% insulin, 10% fetal bovine serum,and 0.1% 1000×gentamicin/amphotericin-B. Upon thawing, cells areresuspended in FGM3 and seeded at −3.5×10³ cells/cm². After reaching 80%confluence, cells are rinsed with 1×PBS and incubated with 0.25%Trypsin-EDTA (Thermofisher) for 3-5 minutes at 37° C. After cells beganto detach, 2 mL Trypsin Neutralizing Solution (Lonza) are added per mLof TrypsinEDTA, and the cell suspension is centrifuged at 220 G for 5minutes. The media supernatant is aspirated, and cells are eitherre-suspended in FGM3 and passaged at a density of 3.5×10³ cells/cm² orused for casting MIFIs.

C2Cl2s are cultured according to manufacturer specifications. Typically,cells used in experiments are kept below passage 12 and below 80%confluence while in continuous culture. C2Cl2 cells are kept in growthmedia (GM) consisting of high glucose DMEM (Corning) supplemented with1% 100×Penicillin-streptomycin (Thermofisher), 1% 100×L-glutamine(Thermofisher), and 10% fetal bovine serum (JR Scientific). Generally,when cells reach 80% confluence, the cells are passaged by washing with1×PBS and incubated with 0.05% Trypsin>EDTA for 3-5 minutes. Cells arethen resuspended in GM at a 2:1 ratio of GM to Trypsin-EDT A andcentrifuged at 2000 RPM for 5 minutes. The supernatant is aspirated fromthe cell pellet, and cells are reseeded at ˜6.5×10³ cells/cm² in a newflask or cast in MIFI construct.

Prior to tissue culture, PDMS wells are cleaned by sonicating in 50%ethanol for 30 minutes. The PDMS strips and vacuum grease can beUV-treated for 15 minutes prior to use. PDMS wells can be dried using anitrogen air gun and then incubated with 1% w/v Pluronic F-127 (Sigma)for 3-5 minutes to prevent cell attachment to PDMS. Typically, PluronicF-127 is aspirated and wells are rinsed 3 times with 1×PBS. The strips(e.g., strips 112) are then placed in the wells by securing both ends inslits (e.g., slits 106 a-b of FIG. 1A) in the base of the PDMS wells.Before strip placement, the bottom of the PDMS well can be coated withvacuum grease, and wells are then firmly placed in the well of a cultureplate. The culture plate used in experimentation was a 6-well plate.However, well plate size include other sizes, such as a 48 well plate ora 96 well plate. Wells with strips are then used immediately orsterilely stored for later use.

For experimentation purposes, rat tail Col I (Corning) was gelledfollowing the neutralization reaction using IN NaOH (Sigma) asrecommended by the manufacturer. MIFIs may be generated using multiplecollagen concentrations. In one example they are cast with finalconcentrations of 2 mg/mL (e.g., 2, 5, 10, etc. mg/mL) Collagen I, 20%v/v Matrigel™, 10% 1×PBS (Hyclone), 2.3% 1 NNaOH, and 18.75×10⁶ cells/mLfor CMs and 15×10⁶ cells/mL (or other similar concentrations) forC2Cl2s. Other materials (e.g., fibrin) can be substituted for the cellculture. Additionally, the concentrations of cells can be increased ordecreased from these values, which can be is done to tune tissuedensity. When NIICV-Fs were included with HUES9-CMs, they were mixed atratios of 10 or 20% of total cell concentration. After manual mixing,the cell/gel mixture was pipetted gently into wells (80 μL/well) toavoid creation of bubbles. To prevent MIFIs from coming out of the wellsdue to vigorous beating at later dates in culture, No. 2 55×45 mm coverglasses can be cut into rectangles <3 mm in width and at least 10 mm inlength using a diamond tip pen, inserted on top of PDMS wells but understrips and secured with vacuum grease. The MIFIs were placed in a 37° C.incubator for 45 minutes to allow gelation of the Col I prior toaddition of culture media. CMs were cultured in RPMI 1640 with 1%knock-out serum replacement (Thermofisher) and 1:000 Mycozap. In someimplementations, C2Cl2s are cultured in GM for 1 week before switchingto DM. CM and C2Cl2 media can be exchanged every 48 hours. Thisparticular process can vary depending on the cell culture beingdeveloped. MIFIs were kept in culture up to 14 days before removal fromwells for the contractility assay.

MIFIs can be imaged on a stereo microscope with oblique illuminationduring culture for top down images of tissue area. Images are convertedto grey-scale, thresholded, and then converted to an 8-bit binary inorder to distinguish the tissue area from the rest of the well. Thepercent area of the tissues was tracked in this way for the duration ofculture.

MIFIs were removed from wells using forceps and were transferred to a 35mm petri dish filled with 37° C. Tyrode's solution (1.192 g HEPES, 0.203g MgCl₂, 0.403 g KCl, 7.889 g NaCl, 0.04 g NaH₂PO₄, 0.901 g C₆H₁₂O₆, and0.265 g CaCl₂) per liter of distilled water, pH 7.4). MIFIs wereanchored by placing the wide base of the strip into a slit in a PDMSblock glued to the bottom of the stimulation dish. The Tyrode's bath wasmaintained between 30° and 37° C. using a heated stage regulated by anin house Lab VIEW program. Videos of samples were taken using a NikonD5100 DSLR camera mounted on a Nikon SMZ1500 stereomicroscope. Sampleswere paced from 2-10 Hz using parallel platinum electrodes placed 2 cmapart and immersed in Tyrode's solution. Samples were stimulated with40V using a 10 ms square pulse wave. Shorter pulse durations can also beused (e.g., 2 ms, 4 ms, 6 ms, etc.).

Carbachol and isoprenaline were mixed to stock concentrations of 5 mM in1×Tyrode's solution. 5 mM epinephrine stock solution was made bydissolving epinephrine at 500 mM in HCl before diluting with Tyrode's toa final concentration of 5 mM. Caffeine was dissolved directly inTyrode's to make a stock solution of 20 mM. All stock solutions werediluted further with Tyrode's to reach concentrations of 50 nM, 500 nM,5 μM, and 50 μM for carbachol, isoprenaline, and epinephrine. Caffeinewas used at 500 μM or 5 mM concentrations. These concentrations wereused because this range had been previously demonstrated to elicitchanges in force exertion and beats per minute (BPM) from cardiactissues in a dose-dependent manner. During the contractility assay,MIFIs were washed 3× with Tyrode's between drugging conditions, andvideos of constructs in Tyrode's only were taken between different drugconditions to determine if the MIFI had recovered to its initial,predrugged state.

Samples were fixed in 4% formaldehyde (electron microscopy sciences) inPBS with 1:200 Triton-X 100 (Fisher Scientific) for 1 hour followed bythree 30 minute washes in PBS. Samples were blocked in 5% goat serum inPBS overnight at 20° C. followed by three 30 minute washes in PBS. Mouseanti-sarcomeric-α-actinin (Sigma-Aldrich) was diluted to 1:100 in PBS,and samples were incubated overnight at 20° C. in 500 μL of antibodysolution in 24 well culture plates to ensure coverage of the 30 tissues.Samples were then washed 3 times for 30 minutes each in PBS beforeincubating with 1:200 DAPI, 3:200 phalloidin tagged with Alexa-Fluor488, and 1:1000 goat anti-mouse antibody tagged with Alexa-Fluor 555 inPBS at 20° C. overnight. After the final incubation step, samples wererinsed 3 times for 30 minutes each in PBS before imaging or storing inPBS.

Finite element modeling of bending PDMS strips was used to create lookuptables for strips ranging in thickness from 125 to 154 μm. FIG. 9 showsmodeling data 930 for strips 900, 910, and 920. The elastic strip (e.g.,strip 112) was modeled as 3D deformable extruded solid, with extrudedthickness varying between 125 and 154 μm. The elastic strip wasconsidered as linearly elastic, isotropic and incompressible material,with Young's modulus equal to 3.09 MPa according to elastic modulusmeasurements. The section of the elastic strip was considered as solidhomogeneous. The force-driven bending simulation was designed to mimicthe experimental procedure. One static general analysis step was usedwhere the elastic strip was deformed by application of equal contractileforce in three parallel axial connectors placed in the regions where thetissue was attached during the experiment, so that the total force wouldequal the sum of the contractile force of the three connectors. Thetotal force ranged between 60 and 155 μN for the experimentally measuredtissue length that corresponded to the deformed length of theconnectors. Additional boundary conditions were imposed based ongeometrical symmetry. The general analysis step did not have automaticstabilization applied, and the step increments were assignedautomatically. Finally, the finite element mesh was composed of 20-nodequadratic, hybrid, reduced integration brick elements. From this model,the change in tissue length could then be used to calculate the forcenecessary to bend the strips to these conformations. Length changes ofcontracting MIFIs were tracked, and resulting length data was used tocalculate the corresponding force based on tissue length and stripparameters (e.g., thickness, width, length, elastic modulus, and shape).

To approximate tissue cross-sectional area to normalize measured twitchforces, microtissue width and thickness were measured usingimage-processing techniques. Specifically, width and thicknessmeasurements were taken per construct, one within 1 mm of either stripattachment site and 1 mm from the middle of the tissue. Tissues wereassumed to have an ellipsoid cross-section, and these width andthickness measurements were used as the long and short diameters of thecalculated ellipsoid cross-section. Additionally, muscle cross-sectionalarea was approximated to normalize force per muscle area by assumingfull cell coverage within the outer 100 μm diameter of constructs.Muscle area was then calculated as 90% or 80% of full area for 10% and20% FB tissues, respectively. In this way, muscle cross-section waslikely over estimated, but this allowed for the normalized force to bean underestimation for a worst case scenario. Specific force wascalculated by dividing twitch force by the approximate cross-sectionalarea and for approximate muscle cross-sectional area for each individualtissue.

ANOVA on ranks was performed on data presented in FIG. 6, FIG. 14, andFIG. 16 graph 1640 using the Dunn's method posthoc test, and ANOVA onranks with post-hoc Tukey test was performed as shown in FIG. 16 graph1650. Two-way ANOVAs with Holm-Sidak post-hoc comparisons were performedfor FIG. 16 graphs 1600, 1610, and 1620. A Mann-Whitney Rank Sum testwas performed on FIG. 16 graph 1630. A One-Way ANOVA was performed ondata in FIG. 14 graph 1410 with no post-hoc test as no significantdifferences were found. Differences were considered significant atp≤0.05. Sample sizes for twitch force graphs in FIG. 14 graphs 1100,1110 represent five technical replicates/sample, i.e. if n=30, then fivebeats/sample for six separate samples are represented.

Returning to FIG. 3, a system for forming contractile, 3D muscle tissuesthat could be easily removed from culture wells and analyzed in a sideon contractility assay is shown.

A U-shaped, thin PDMS strip (e.g., strip 112) is an indicator to trackforce exerted by beating tissues. The strip is designed to have baseshidden in slits in the PDMS culture well and a narrower attachmentaround which the tissue can polymerize, compact, and mature. Asdiscussed in reference to FIG. 1A-2B. This relatively wide strip baseenables eventual removal of the tissue from the well, at which point,contraction of the beating cells induced a change in tissue length andbends the strip, shown at 310, 320 of FIG. 3. In this way, MTFIs wereeasily assessed for overall tissue beat frequency and muscle forcegeneration. Typical 3D construct designs in the past had PDMS posts thatdictated tissue geometry designed as a part of the well itself, andremoval of the tissue from the well would result in a contractile tissuewith no load to work against relative to itself.

Turning to FIG. 4, in order to allow removal of the tissue while stillmaintaining adherence to post-like structures, PDMS molds were designedto have aslit in the base of the well between 2 small posts. U-shaped,thin, PDMS dogbone-like strip configured for being inserted orthogonallyto either end of the tissue during the Collagen I polymerizationprocess, but hidden between the PDMS posts. Initial designs were testedusing C2Cl2 tissues, tissue attachment, but large holes resulted fromthe double post design. To reduce resulting holes in the tissues butmaintain the classical post design, the inner posts are removed and madethe wells made smaller. The resulting MIFIs appeared to have improvedtissue integrity at the attachment sites, shown by 410 g. Aftersuccessfully removing the inner posts for Design 2, we wanted todetermine if posts were necessary or if the PDMS strip alone was enoughof an anchor point on either side of the well for tissues to attach andcompact without any posts. Well 420 was developed to determine tissuescould be cast around the strip alone 420 i-j. C2Cl2 MIFIs attached andcompacted with no apparent issues in well 420; however, the well slitsfor strip placement were wide enough to allow the cell+gel mixture toseep into the slits, which were ˜1 mm wide, before polymerization hadoccurred. This resulted in small tissue overhangs shown in 420 k.Tissues were able to move up the strips since there was nothingrestricting them to the initial attachment sites after removal fromwells 420 k.

FIG. 4 shows an iterative plastic mold and PDMS strip design process. At400 a, a perspective view of CAD model of a plastic mold design isshown, intended to hide the strip between 2 PDMS posts on either side.The mold is the negative of what PDMS wells look like. A cutaway view ofthe dotted black rectangle shows side view 420 b of Design 1 wherearrows point out what will become PDMS base slits and additional arrowspoint out what will become PDMS posts. Image 420 c shows that C2Cl2 MIFIcreated using Design 1 resulted in the possibility of large holes in thetissue around the strip because it was ‘hidden’ between 2 PDMS posts(see arrows). Strip 420 d is a PDMS strip with dimensions for moldDesign I. Image 420 e shows a perspective view of CAD model of plasticmold Design 2, intended to hide the strip against 1 PDMS post on eitherside to decrease hole size in tissues. This is the negative of what PDMSwells would look like. A cutaway view 420 f of the dotted blackrectangle shows a ide view of Design 2 where arrows point out what willbecome PDMS base slits and additional arrows point out what will becomePDMS posts. Image 420 g shows a C2Cl2 MIFI created using Design 2 thathas better tissue integrity around the attachment sites. Strip 420 h isa PDMS strip with dimensions for mold Design 2. The strip length wasshortened to match the shorter well dimensions after removal of innerposts. Image 420 i includes a perspective view of CAD model of plasticmold Design 3, intended to completely replace posts with the strip. Thisis the negative of what PDMS wells would look like. A cutaway view 420 jof the dotted black rectangle shows a side view of Design 3 where arrowspoint out what will become PDMS base slits. Image 420 k shows a C2Cl2MIFI created using Design 3. We observed small overhangs of tissueformed from seeping into the base slits before the Collagen I hadpolymerized and the tissue was beginning to slip up the strip (arrows).The scale bars for 420 c, 420 g are 4 mm. The scale bars for 420 k are 3mm.

In some embodiments, as shown in FIG. 5, PDMS well 500, 510 includes5×10 mm wells and strip slots that are 500 μm wide (to prevent tissueoverhangs) and 3 mm deep. The thin, PDMS strip 520 (e.g., equivalent tostrip 112 of FIGS. 1A-2B) was designed to have a base to sit flushwithin the well slit, a 1.5 mm necking region for the cast tissueattachment, and a small 3.4×1 mm flap above the attachment region toprevent tissue slippage. These strips were laser-cut from pre-made 127μm thick PDMS sheeting, and the final PDMS well with strips placed inallowed for the base of the strips to sit flush against the well slitportion, shown in in images 550, 560, and 570.

Image 500 shows a top down view of the dimensions of the raised portionof the plastic mold used for casting wells for MIFIs. Smaller rectangles(arrows) served as the strip slots in the PDMS well. Image 510 shows thefinal design of the well (e.g., well 100), which had a base with 3 mmdeep slots to ‘hide’ strip base in the PDMS well during culture. Image520 shows a preferred strip design (e.g., strip 112) included 3 mm×3.4mm regions to be hidden in the PDMS well base, a 1 mm wide neckingregion for tissue attachment, and a small 3.4 mm wide overhang toprevent the tissue from sliding up the strip. Image 530 shows a top downimage of the plastic master mold and image 540 shows resulting PDMSwells. Images 550, 560, and 570 are side, top, and perspective views,respectively, of the strip placed in the final well design. The stripcan be seen resting flush against the in the PDMS base slits (arrows).All dimensions are in mm.

Turning to FIG. 6, day 1 images 600 a-I show constructs cast withHUES9-CMs and 10% FBs (600 a), 20% FBs (600 b-c), 0% FBs (600 d).Further shown are day 10 images of 10% FBs (600 e), 20% FBs (600 f-g),and 0% FBs (600 h), which only show visibly more compact tissues intissues seeded with FBs compared to HUES9-CMs only. ˜30% of tissues with20% FBs exhibit irregular remodeling and break away from the strips asseen in image 600 g. 10% FB (image 600 i) tissues continue to uniformlycompact at day 14 in culture. Graph 600 j details this phenomenon andincludes plots of tissue compaction over time, as represented by percentof the original area, show that both 10% and 20% PB tissues have slowedcompaction rates after the first three days in culture. Tissues with 20%FBs have less uniform compaction compared to 10% FB tissues, as shown bylarger standard deviation bars associated with these tissues. Graph 600k shows Day 10 of compaction area, as represented by percent of originalarea, is significantly affected by the addition of FBs. Dotted blacklines outline the tissues and scale bars are 2 mm.

In an initial study to determine if Col I gels with HUES9 derived CMsinduced bending of PDMS strips, tissues with CMs only were cast in theprototyped well design and allowed to culture for 10 days after casting,shown in images 600 a,d,c,d. While contraction of MIFIs induced bendingof strips, the CM-only tissues did not significantly remodel thesurrounding Col I gel. This was problematic as these tissues arediffusion limited to having viable cells within the outer 100 μm of gel,and the initial tissue dimensions (˜1×10×5 mm) were an order ofmagnitude outside of the diffusion limits of non-vascularized tissues.

In order to engineer more cell-dense MIFIs that compacted over time, weadded cardiac ventricular FBs to assist the CMs in remodeling thesurrounding Col I+Matrigel™ mixture, a method that had been previouslyshown to work in 30 engineered cardiac muscle. FB populations used inthe literature were derived from varying sources and may behavedifferently than the commercially available FBs we used, so we added 10%or 20% FBs to MIFIs as ranges of 3%-30% FBs had been reported aseffective. We found that the addition of FBs resulted in visualcompaction of the MIFIs, and representative images of those with 10% FBsat Days 1, 10, and 14 showed relatively uniform compaction as far as 2weeks in images 600 a, 600 e, and 600 i. For 20% FB MIFIs some tissuesdisplayed uniform compaction by Day 10 in culture shown in images 600b,f, but as many as 50% of constructs tore due to less uniformcompaction by the higher FB population shown in images 600 c,g.Compaction area of these tissues were tracked from Day 0, when all wellswere 100% filled (by area), to Day 10 or 14, depending on FBcomposition. Both FB concentrations resulted in compaction to ˜50% ofinitial area by Day 3 after casting (10% FB—52.2%, 20% FB—46.0%) shownin graph 600 j. By Day 10, 20% FB MIFIs had compacted to ˜10% less area(27.8%±6.62) than 10% FB MIFIs (38.5%±3.88), but as previouslymentioned, these tissues were less uniformly compacted. 10% FB MIFIswere easily maintained in culture for at least 14 days, when theyreached 31.7%±3.26% of their original area shown in graph 600 j. Thus,while 20% FB MIFIs were significantly more compacted by Day 10 than the10% and 0% FB MIFIs, the compaction was less predictable and oftenresulted in up to 50% sample loss due to breakage graph 600 k. The 10%FB MIFIs were able to compact significantly more than 0% FB MIFIs 600 kand were able to be maintained for longer culture times due to moreuniform compaction by the smaller FB population.

MIFI with 10% and 20% FBs were fixed and stained for sarcomericα-actinin, F-actin, and nuclei to qualitatively observe if more spread,striated CMs were observed as length of culture time increased (FIG.7A-7B). Generally, 10% FB MIFIs appeared to have more spread cells atall time points compared to 20% FB MIFIs, as observed by F-actinstaining across samples. Additionally, 10% FB MIFIs did not appear to beout-populated by the FB subpopulation of cells, as most tissues hadcells with well-spread regions of α-actinin throughout, and striationswere observed in these CMs as early as 7 days in culture. Tissuesappeared to have sarcomeres that were more aligned in the direction ofthe long-axis of the MIFI as culture time was extended to 14 days. 20%FB MIFIs appeared to have less CM coverage until Day 10 of culture, butin these samples, sarcomeres did not appear to organize along thelong-axis of the MIFIs as much as was observed in 10% FB MIFIs.

FIG. 7A shows meso-scale images 700 of CM tissues with 10% FBs stainedfor nuclei, actin, and sarcomeric α-actinin and that appear to showincreased cell spreading as tissues were kept in culture over longerperiods of time. Representative microscale images of features of thesame tissues show that cells appear well spread for all time points, butthe differences observed in meso-scale and microscale images at Day 10compared to Day 7 and Day 14 may result from remodeling within tissuessince visible beating was observed in wells beginning around Day 7.Sarcomeric α-actinin staining overlaps with most actin staining, showingthat FBs are not overpopulating the tissues and CMs were the major celltype at all time points. Finally, zooming in on the microscale imagesshows striations are present in all of the μtissues, and they appear tobecame more organized and uniform over time (light arrows). Dark arrowsrepresent a long axis of the tissues.

FIG. 7B shows meso-scale images of CM MIFIs with 20% FBs stained fornuclei, actin, and sarcomeric α-actinin appear to show increased cellspreading as tissues are kept in culture over longer periods of time.Representative microscale images of features of the same tissues showthat cells were not as well spread at days 6 and 8 for 20% FB tissuescompared to 10% FB tissues at Day 7. Sarcomeric α-actinin staining didnot appear as dense at Day 6 and Day 8 tissues compared to Day 7, 10% FBtissues. However, zooming in on the microscale images showed striationswere present in all of these tissues as well (light arrows). Dark arrowsrepresent long axis of tissues.

Turning to FIG. 8 collagen I structure and cell organization are shown.Image 800 is a top down image of second-harmonic signal of Col I infixed tissue with 10% FBs after 10 days in culture shows some alignmentin the direction of the long axis of the tissue (represented by whitearrows). Image 840 shows a cross-sectional view of Col I shows it ismore densely packed in the outer 100 μm, likely due to cellularremodeling in this region. The cross section only shows half of thetissue; the Col and cells caused significant light scattering, andclearing methods will need to be used in the future to analyze fulltissue cross-section and structure. Images 810, 830 show representativetop down and perspective images of a day 10, 20% FB tissue stained fornuclei, actin, and α-actinin. A more densely packed middle regionappears to be aligning parallel to the long axis of the tissue,resulting in a less elliptical tissue cross-section, a phenomenonobserved in several of the 20% FB tissues.

The second-harmonic generation of Col I was used to observe the Col Istructure in 10% FB MIFIs. The cells and Col I were densely packed inthe outer 100 μm of MIFIs, so imaging the Col I structure past 400 μmwill not be possible until clearing protocols are implemented to reducelight scattering and allow imaging through the full tissue thickness.Unsurprisingly, cells were generally restricted to the outer 100 μm ofthe MIFIs due to diffusion limits in these unvascularized tissues.Interestingly, in several of the 20% FB MIFIs at Day 10 of culture, thecells remodeled the middle region of the MIFI to be more compact andaligned with the long axis of the MIFI. It is possible that thisless-uniform remodeling was also responsible for the breakage of 30-50%of MIFIS/trial that was observed in 20% FB samples.

Overall, striated CMs were found in the MIFIs after Day 6 for both 10%and 20% FB tissues. 10% FB MIFIs appeared to have better spread CMs atlater time points and had more evenly distributed CMs at Days 7 and 10compared to %20 FB MIFIs. Unsurprisingly, tissues had a ‘dead zone’ 100μm deep into the tissues, likely because cells migrated to the outerregion of the gel and began remodeling from the outside-in. However,more analysis needs to be done on cell-orientation, more precisecell-composition at different time points, and improved clearing orimaging methods must be implemented to further characterize theseaspects of the engineered MIFIs.

FIG. 9 shows images 900, 910, 920 of finite element modeling of abending PDMS strip 125 μm thick with the tissue modeled as connectors(dotted black lines) attached to either side of the strip. Images 900,910, 920 represent strip bending under tissue loads of (A) 65 μN, (B) 75μN, and (C) 85 μN, respectively. Plot 930 includes curves used togenerate lookup tables from tissue length. Specifically, tissue lengthis plotted against and force required to bend the strip to saidconformation, and each line represents a separate lookup table for stripthicknesses in 1 μm increments (125-150 μm and 154 μm).

Finite element modeling of bending of the thin, PDMS strips was used togenerate lookup tables that relate tissue length and force required toinduce bending of a strip to that conformation. Specifically, the stripswere modeled as having connectors (or the tissue) attached at eitherside of the necking region of the strip shown in image 900. As thecompressive force required to bend the strip increases, the connector(or tissue) length decreases, as shown in images 910, 920. In this way,plots of the relationship between tissue length and force exerted onPDMS strips were generated for strip thicknesses ranging from 125-154μm, as shown in graph 930.

Turning to FIG. 10, a well plate 1000 is shown. The well plate can beused for high-throughput assays of tissue samples. Each well 1002 of thewell plate 1000 includes a tissue-strip configuration as described above(e.g., well 100, strip 112, and tissue 200 of FIGS. 1A-2B). For example,each well has a strip 1004 that exerts a stress on the tissue, thestress being tuned to a particular value based on adjusting one or moreparameters (e.g., length, width, thickness, elastic modulus, shape,etc.) of the strip. Each of strips 1004 can be tuned with pre-definedparameter values. The well plate 1000 can be configured to test amultitude of permutations of tissue compositions with different stresseson those tissues 1006. The well plate 1000 can form an N×M array oftissue samples, with N rows and M columns. In some implementations, eachrow or column (or both) can include strips 1004 with particularparameters that are identical to one another, and different tissuecompositions, densities, etc. can be tested. In such a setup, eachtissue sample 1006 a-n and/or 1006 a-m can be combined with a compound(e.g., a drug compound) to test the effects of the drug on the tissue1004 a-m. In some implementations, each well 1002 a-m can include aunique combination of tissue composition and/or strip 1004 parameters.The strips 1004 a-m and/or the tissues 1006 a-m thus form a parameterspace, including a parameter space of stress values by the strip (e.g.,as a result of adjusting parameters of the strip), and/or a parameterspace of tissues (e.g., by adjusting tissue composition, density, etc.),and/or a parameter space of compounds (e.g., drug compounds) beingtested on the tissues 1004.

For example, each tissue 1004 a-m can be injected with a compound, andthe results measured for the respective tissue 1004 a-m. The resultsmeasured can be based on the assay performed, and can include any of theeffects mentioned herein, such as twitch force, contraction percentage,contractile force, etc. For example, the tissues can be introduced toany drug compound, including caffeine, isoprenaline, ephedrine,amphetamine, etc. In some implementations, a muscarinic agonist can beintroduced (e.g., carbachol, pilocarpine, oxotremorine, etc.). The wells1002 a-m, strips 1004 a-m, and tissues 1006 a-m of well plate 1000 arethus useful for drug screening methods, because they provide a highlytunable tissue (particularly regarding contractile variability) thatexhibits adult-like physiology.

FIG. 11 shows a process 1100 for performing a non-invasive contractilityassay of a tissue (e.g., tissue 200 of FIGS. 2A-2B). The method includesgenerating (202) a tissue that is affixed to a strip of bendablematerial. The tissue is affixed to a first end and a second, oppositeend of the strip as described above. The method includes causing (204)the tissue be in a contracted state and exert a stress on the strip tobend the strip. The tissue exerts a contraction force on the strip asdescribed in relation to FIGS. 2A-2B. Here, causing the tissue to exerta stress force on the strip includes generating the tissue such that thetissue connects the first and second ends of the strip and causes thestrip to bend. In addition to the contractile force of the tissue,additional contraction forces can be induced by stimulating the tissuewith electrical signals. When the tissue is contracting (either inresponse to the electrical stimulation or when the tissue is removedfrom the well), the curvature of the strip is measured (206). Thecurvature can be measured by image processing or other such techniques,as described in relation to FIG. 20. Measuring the curvature can includemeasuring an area under the curve of the strip, or other metrics formeasuring the curvature can be used. The method includes calculating(208) the stress exerted on the strip by the tissue, the stress being afunction of the curvature of the strip and one or more parameters of thestrip, the one or more parameters each having a value that ispre-determined. The stress is a function of the curvature of the stripand the known quantities of the thickness, material type, elasticmodulus, width, length, shape, etc. of the strip. For example, for aknown curvature of the strip, the stress that the strip is exerting onthe tissue can be calculated. From the calculated stress of the strip onthe tissue, the responsive contractility of the tissue can bedetermined.

FIG. 12 shows a process 1200 for generating a generating a threedimensional tissue with an integrated load. The process 1200 includesadding (1202), to a well (e.g., well 100 of FIG. 1, 1002 a-m of FIG. 10,etc.), a cell suspension mixture, the well comprising a strip ofbendable material, wherein the strip of bendable material is insertedinto the well at a first end and at a second end opposite the first endso that the strip is curved (e.g., as seen in FIGS. 1A-1B). The process1200 includes generating (1204), from the cell suspension mixture, atissue that is affixed to the first end of the strip and the second endof the strip. The process 1200 includes removing (1206) the strip fromthe well, wherein the strip is configured to exert a stress on thetissue after the strip is removed from the well, and wherein the stripexerts a reduced stress on the tissue before the strip is removed fromthe well relative to an increased stress on the tissue after the stripis removed from the well. The strip is configured to exert a reduced(e.g., non-maximum) amount of stress on the tissue before the tissue isremoved from the well. In some implementations, the strip exhibitsminimal/negligible stress on the tissue before the strip and tissue areremoved from the well. The strip is configured to exert a maximum (e.g.,unimpeded) stress on the tissue after the strip and tissue are removedfrom the well because the strip is no longer braced by the well.

Turning to FIG. 13, a schematic 1300 shows a configuration of a side-oncontractility assay for MIFIs. MIFIs were removed from wells for thecontractility assay at Day 7, 10, or 14 (10% FBs) or at Day 6, 8, or 10(20% FBs). By tracking the change in tissue length of beating MIFIs, wecalculated force generated by these tissues using force lookup tables.MIFIs were removed from wells, one side of the strip was placed in aslit in a PDMS block to anchor the MIFI for side-on viewing, and theconstruct was submerged in Tyrode's and placed between two parallelplatinum electrodes for later stimulation by assay device 1300. MIFIsspontaneously beat, resulting in visible deformation of the PDMS stripwhen imaged in the side-on contractility assay. Day 14, 10% FB MIFIsexerted significantly higher twitch force (3.62±1.7 μN) compared to 20%FB tissues at Days 6, 8 and 10 (1.03±0.70 μN, 1.65±1.4 μN, and 0.82±0.45μN respectively). These tissues also beat with higher twitch force thanDay 10 MIFIs with the same FB percentage (0:773±0.55 μN), shown in graph1400 of FIG. 14. Day 7 MIFIs with 10% FBs also exerted higher twitchforces (2.44±1.3 μNT) than Day 10 MIFIs with 10% or 20% FBs. It ispossible that this decrease in contractility at Day 10 was a result ofremodeling in these tissues around Day 10, something that was reflectedin the fluorescence staining. Because MIFIs began visibly beating in thewells by Day 7, it is possible that this induced the CMs and FBs toremodel the surrounding matrix at a mesoscopic level.

Tissues also responded to electric field stimulation and were capable ofbeing electrically paced. When twitch forces of MIFIs paced at 2 Hz weremeasured, Day 14, 10% FB MIFIs exerted higher twitch force (3.07±1.54μN) compared to Day 6, 8, and 10 MIFIs with 20% FBs (1.08±0.52 μN,1.61±0.89 μN, and 0.93±0.426 μN). It appeared that, overall, 20% FBMIFIs did not exert as much force as 10% FB MIFIs. In part, this is dueto earlier testing dates of the 20% tissues because these tissues werenot able to be stably maintained in culture as long as the tissues with10% FBs. Thus, these MIFIs had less time to mature and reorganizematrix, and the FBs in these tissues overpopulated the MIFIs andprevented, CMs from synchronizing throughout the tissue. Constructs atall time points were assessed for spontaneous beat frequencies whichranged from 60 BPM±15 (10% FB, Day 7) to 107 BPM±38 (20% FB, Day 10).However, no significant differences were found between groups sinceMIFIs at these time points native beat frequencies are still variable,something that had been observed in individual wells of differentiatingCMs as well.

As stated above, FIG. 13 shows a schematic 1300 of the side-oncontractility assay for MIFIs. The microscope objective views the top ofthe set up. The MIFI is placed on its side, with one strip anchored in aslit in a PDMS block glued to the bottom of the petri dish. The rvrIFIis submerged in Tyrodels and placed between two parallel platinumelectrodes for electrical pacing. Side on images of cardiac MIFIs in thecontractility assay image 1310 systole and image 1320 diastole, with theyellow dotted line showing the change in tissue length. Scale bars are 4mm.

In FIG. 14 graph 1400 represents twitch forces (systole-diastole) forspontaneously beating tissues and shows that 10% PB tissues at Day 14had significantly higher twitch forces than Day 10 samples with 10% FBand 20% PB samples at all time points. Day 7, 10% FB tissues also hadsignificantly higher spontaneous twitch force than Day 10 samples with10% or 20% FBs. Graph 1410 shows twitch forces for electrically paced (2Hz) samples which showed similar trends, with Day 14, 10% FBs exertingsignificantly higher twitch force than Day 10, 10% and 20% FBs and Day6, 20% FB samples. Twitch force appears to decrease slightly uponelectrical stimulation, but variation in resulting forces decreases.Graph 1420 shows spontaneous beat frequencies (in BPM) of cardiactissues with 10% or 20% FBs analyzed at Day 7, 10, 14 or Day 6, 8, and10 in culture, respectively. No statistically significant differenceswere found among samples with n less than or equal to 3, with meanvalues ranging from 60 to 107 BPM (Day 7, 10% FBs and Day 10, 20% FBs,respectively).

Turning to FIG. 15, example force vs. time plots are shown of a Day 14,10% FB MIFIs demonstrate a regular beat frequency and the ability toelectrically pace the samples at 2 Hz 1500 a. Titrating in isoprenaline,an excitatory drug, had the intended stimulatory effect on the nativebeat frequency of MIFIs at 500 nM and 5 μM shown in graphs 1500 b,1500c, but it did not interrupt the ability to electrically pace theconstructs at these concentrations. The addition of isoprenaline did notnoticeably affect the twitch force of this specific MIFI at theseconcentrations, which exerted twitch forces ˜4.6 μN when beatingspontaneously in Tyrode's and ˜4.0 μN when beating spontaneously in 5 Misoprenaline. Washing out the isoprenaline dosage with Tyrode's recoversthe MIFI to its initial beat frequency, shown in graph 1500 d.Subsequent treatment with 55 mM caffeine also increased the spontaneousbeat frequency with minimal impact on the twitch force, and dosing withthe muscarinic agonist, carbachol, reduced the spontaneous beatfrequency while having surprisingly little impact on twitch force, asshown in graphs 1500 e-f.

While most MIFIs beat more synchronously as culture time increased,occasionally disease-like arrhythmic beating was observed in these Day14 construct, seen in image 1500 g. While this disease-like constructwas still able to be electrically paced, its native and paced beatingplots of force reflect the abnormal beating, especially when compared tomore synchronous constructs such as shown in graph 1500 a. To determineif the MIFI could recover to a more normal beating phenotype, increasingcarbachol doses were administered (500 nM, 5 μM, and 50 μM) and resultedin improvement in the spontaneous beat frequency of the constructs, butthe twitch force profiles were still ˜30% the strength of the twitchforce of synchronous constructs, shown in graph 1500 h. The arrhythmicconstruct was electrically paced at doses of 5 μM and 50 μM ofcarbachol. Unlike electrical stimulation of the construct with no drugtreatment graph 1500 g, the combinatorial effect of carbachol treatmentwith electric pacing at 2 Hz resulted in significantly higher twitchforces and a normal phenotype beating waveform was observe, such asshown in graph 1500 i. More interestingly, a significant interactionbetween the stimulation and drug treatment was found during 2-way ANOVAanalysis of twitch force under these conditions shown in graph 1600 ofFIG. 16. 2-Hz pacing significantly increased twitch force with orwithout drug treatment, and pacing coupled with 5 or 50 μM carbacholresulted in 2.5× and 2.8× stronger twitch contraction, in graph 1600.

Turning to FIG. 16, additional analysis of specific specimens of Day 14,10% FB MIFIs showed decreasing twitch force as stimulatory drug dose wasincreased. Specifically, one MIFI exerted 3.54 μN±0.12 spontaneoustwitch force and 3.34±0.935 twitch force under 2 Hz pacing with no drugtreatment, shown in chart 1610. Dosing with 500 nM isoprenaline resultedin a drop to −80% of initial twitch force to 2.85 μN±0.84 and 2.81μN±0.71 for spontaneous and 2 Hz paced beating, respectively. Increasingdrug dosage to 5 mM resulted in an even further decrease (1.78 μM±0.21)to ˜50% of initial twitch strength, and electric pacing at 2 Hz wasunable to recover full twitch strength (2.15 μN±0.33). Epinephrinetreatment of a separate MIFI construct also resulted in a dose-dependentdecrease in twitch force, shown in graph 1620. Specifically, duringspontaneous beating, no drug treatment and 500 nM drug treatmentresulted in 5.93 μN±0.44 and 5.49 μN±0.25 twitch force respectively.These values dropped to 66% and 61% of the initial twitch force at 5 μMand 50 μM epinephrine doses. Interestingly, two-way ANOVA analysis foundthat, the effect of electrical stimulation was significantly higher onlyas dosage of epinephrine increased. The twitch forces at 5 μM and 50 μMwere significantly recovered by electrical pacing back to 85% and 80% ofinitial twitch force, respectively. While it is not initially intuitivethat some stimulatory drugs resulted in decreasing twitch force asdosage increased, it is possible that this was a result of increasedbeat frequency of the constructs, resulting in an increased diastolicforce between beats. This hypothesis is also supported by the evidencethat pacing constructs at 2 Hz significantly improved the twitch forceof dosed constructs, shown in graphs 1600 and 1620.

The magnitude change in BPM was compared, and each MIFI was normalizedto its native beat frequency due to the wide range of beat frequenciesshown in all constructs. MIFIs dosed with increasing concentrations ofisoprenaline beat at 1.2 1×±0.2, 1.27×±0.10, and 1.48×±0.47 initial beatfrequency for 50 nM, 500 nM, and 50 μM treatments, respectively, shownin graph 1640. Epinephrine dosing similarly affected MIFI beatingfrequencies by raising it to 1.42×±0.51 and 1.65×±0.47 initial BPM,shown in chart 1650. Finally, caffeine dosing at 5 mM significantlyincreased BPM to 1.59×±0.451 initial BP, shown in chart 1630. Thus, acardiac tissue was engineered by co-culturing HUES9-CMs and FBs andintegrating a U-shaped PDMS strip, and MIFIs induced visible bending ofthe strip and responded as expected to pharmacological stimuli.

Turning to graph 1700 of FIG. 17, a fuller characterization of MIFIs wasmade to calculate the force measurements per cross-sectional area of thefull tissue section as well as the muscle-only cross-section, a stepthat is necessary to determine if the actual muscle cells in specifictissues are stronger at specific time points. Average tissue width andthickness were measured to approximate an ellipsoidal cross-section toperform initial normalization calculations. The ellipsoidalcross-section was measured by measuring 3 points per construct, and thedistribution of calculated cross-sections had higher variability in the20% FB constructs, which is reflective of the less-uniform compactionobserved in those tissues.

Turning to FIG. 18, graphs 1800, 1810 show that when each MIFI wasnormalized to its average ellipsoidal cross-section, D14, 10% FB MIFIswere found to exert higher specific force (med=1.1 Pa) compared to 20%FB MIFIs at D6 (med=0.45 Pa), D8 (med=0.56 Pa) and D10 (med=0.29 Pa) andcompared to D10,10% FB MIFIs (med=0.30 Pa). D7 10% FB MIFIs exertedhigher specific twitch force (med=0.85 Pa) compared to D10 MIFTs forboth 10% and 20% FB constructs. To further approximate the actual forceper muscle cross-section, the outer 100 μm of MIFIs were assumed tocontain cells, based on initial imaging and the diffusion limits ofnon-vascularized tissues. The outer, cell-containing region wasestimated at composing of 90% CMs for 10% FB constructs and 80% CMs for20% FB constructs to calculate the normalized force per musclecross-section.

Using these approximations, D14 10% FB MIFIs was determined to exertsignificantly higher specific force per CM area (med=12.0 Pa) comparedto D6 and D10 20% FB MIFIs (meds=4.8 Pa and 3.7 Pa, respectively) andcompared to D10 10% FB MIFIs (med=1.9 Pa), shown in graph 1810. D7 10%FB MIFIs exerted higher forces per approximated muscle cross-section(med=9.4 Pa) than D10 10% FB constructs (shown in graph 1810). This dipin force at D10 for 10% FB constructs may have occurred because oftissue remodeling and cell reorganization that occurred after CMs beganto visibly deform the strips in the wells after Day 7. More importantly,these approximations to normalize force for tissue cross-section and CMcross-section still exhibit that constructs are stronger by D14 ofculture, and 10% FB constructs have more uniform remodeling compared to20% FB constructs.

FIG. 20 shows an example of image processing of an example tissue 2010.Image 2000 includes an unprocessed image of a strip 2002 (e.g., strip112) affixed to a tissue 2010 (e.g., tissue 200). The strip 2002 exertsa load on tissue 2010. The tissue provides a contractile force thatcauses the strip 2002 to bend. The amount of force that the tissue 2010is exerted is determined in a non-invasive manner by taking an image ofthe tissue (such as image 200) and determining the curvature of thestrip 2002 in the image 2000. In some implementations, the area 2006inside the tissue 2010 and the strip 2002 is measured to determine thecurvature of the strip 2002. The image 2000 is image processed (e.g., bycolor thresholding, line extraction, etc.) to extract the tissue 2010and the strip 2004 locations in the image. Image 2010 is an example of aprocessed image, where the features of the tissue 2010 and the strip2004 have been extracted. Area 2006 is calculated and used to determinethe curvature of the strip 2004. The curvature of the strip 2004 is usedin combination with the known parameters of the strip (e.g., width,thickness, length, elastic modulus, shape, etc.) to determine what loador stress is being exerted on the tissue by the trip. The determinedstress value is used to calculate the contractile force exerted by thetissue. The image 2000 can be taken when the tissue is static or whenthe tissue is being stimulated (e.g., by an electrical signal).

Various inventive features of a system for generating 3D tissues withintegrated loading have been described above. It will be appreciatedthat not all inventive features need be combined in a single embodiment.Rather, some inventive features may be included within other embodimentswithout using other inventive features. It is to be understood, however,that even though numerous characteristics and advantages of the presenttissue generation system have been set forth in the foregoingdescription, together with details of the structure and function of thetissue generation system, the disclosure is illustrative only, andchanges may be made in detail, especially in matters of shape, size andarrangement of parts within the principles of the tissue generationsystem to the full extent indicated by the broad general meaning of theterms in which the appended claims are expressed.

What is claimed is:
 1. A system for generating a tissue, the systemcomprising: a strip of bendable material, the strip comprising: a firstregion in proximity to a first end of the strip for coupling to a tissuecomprising organic material; and a second region in proximity to asecond end of the strip for coupling to the tissue, the second end beingopposite the first end, wherein the strip is configured to bend to alignthe first region with the second region; and a well for generating thetissue, the well comprising: a region for generating the tissue from acell culture; a first slit configured to receive the first end of thestrip and expose the first region of the strip to the tissue generationregion; and a second slit configured to receive the second end of thestrip and expose the second region of the strip to the region of thewell; wherein the first slit is aligned with the second slit to alignthe first region of the strip and the second region of the strip in theregion to enable the tissue to couple to the first region of the stripand to couple to the second region of the strip during generation of thetissue.
 2. The system of claim 1, wherein the well is configured toreduce a stress exerted by the strip from on the tissue duringgeneration of the tissue relative to a maximum stress that the strip isconfigured to exert on the tissue; and wherein the strip is configuredto exert the maximum stress on the tissue when the strip and the tissueare removed from the well.
 3. The system of claim 2, wherein the stripis configured to provide a stress of up to approximately 10,000 kPa onthe tissue when the strip and the tissue are removed from the well. 4.The system of claim 2, wherein a magnitude of the stress exerted on thetissue by the strip is a function of one or more tuned parameters of thestrip, the parameters comprising a length of the strip, a width of thestrip, a thickness of the strip, an elastic modulus of the strip, and ashape of the strip.
 5. The system of claim 1, wherein the tissue isconfigured for contracting between approximately 10%-40% of an initiallength of the tissue.
 6. The system of claim 1, wherein a cellsuspension of the well comprises an approximate mixture of either 0.5 to10 mg/mL Collagen Type I or fibrin, 20% Matrigel, 10% 10× phosphatebuffered saline, and either 18.75×10⁶ cells/mL for cardiomyocytes or15×10⁶ cells/mL for myoblasts.
 7. The system of claim 1, wherein a cellsuspension of the well comprises a concentration of betweenapproximately 10-100×10⁶ cells/mL and fibroblasts comprisingapproximately 10-20% of a total cell count.
 8. The system of claim 7,wherein cells of the cell suspension comprise one of smooth musclecells, skin cells, ligament cells, and tendon cells.
 9. The system ofclaim 1, wherein the well is a part of a multi-well plate.
 10. Thesystem of claim 9, wherein at least one well of the multi-well platecorresponds to a respective strip having particular parameters, andwherein at least one strip and well of the multi-well plate represent aloading value of a parameter space representing loading values for thetissue.
 11. The system of claim 10, wherein the particular parameters ofthe strip comprise an elastic modulus parameter, a thickness parameter,a width parameter, and a length parameter.
 12. The system of claim 1,wherein the tissue comprises one of a cardiac tissue, skeletal tissue,smooth muscle tissue, skin tissue, cartilage, tendon, and ligament. 13.The system of claim 1, wherein the strip comprises one ofpolydimethylsiloxane (PDMS), Teflon film, a polycarbonate film, or anelastomer film.
 14. The system of claim 1, wherein each of the firstregion and the second region of the strip has a narrower width than awidth of a portion of the strip between the first region and the secondregion.
 15. The system of claim 1, wherein the tissue forms striationsin response to a stress exerted on the tissue by the strip.
 16. Thesystem of claim 1, wherein cells within the tissue align in response toa stress exerted on the tissue by the strip.
 17. The system of claim 1,wherein the tissue is configured to undergo a strain of up to 80% by thestrip when the strip and tissue are removed from the well.
 18. A methodof performing a non-invasive contractility assay of a tissue, the methodcomprising: generating a tissue that is affixed to a strip of bendablematerial, the tissue being affixed to a first end and a second, oppositeend of the strip; causing the tissue be in a contracted state and exerta stress on the strip to bend the strip; measuring a curvature of thestrip when the tissue is in the contracted state and exerting the stresson the strip; and calculating the stress exerted on the strip by thetissue, the stress being a function of the curvature of the strip andone or more parameters of the strip, the one or more parameters eachhaving a value that is pre-determined.
 19. The method of claim 18,further comprising: tuning an action potential of the tissue byadjusting the one or more parameters of the strip; applying a voltage tothe tissue; and responsive to application of the voltage, measuring theaction potential of the tissue using calcium or voltage imaging.
 20. Themethod of claim 18, further comprising measuring an organization of cellcytoskeletal components.
 21. The method of claim 18, further comprisingmeasuring an epigenetic change in the tissue.
 22. The method of claim18, further comprising measuring a gene expression of the tissue. 23.The method of claim 18, further comprising measuring a proteinexpression of the tissue.
 24. The method of claim 18, further comprisingcontrolling, during the generating of the tissue, a density of thetissue by adjusting a concentration of a cell culture in a hydrogelmixture.
 25. The method of claim 24, wherein the hydrogel mixtureincludes at least one of fibrinogen, Matrigel, a hyaluronic acidhydrogel, a synthetic hydrogel, or a natural hydrogel.
 26. The method ofclaim 18, wherein at least one of the one or more parameters comprise athickness of the strip, a width of the strip, an elastic modulus of thestrip and a length of the strip, and wherein the method furthercomprises: selecting the value of the one or more parameters to tune amagnitude of a stress exerted on the tissue by the strip to a particularvalue.
 27. The method of claim 18, further comprising: adding a compoundto the tissue so that the tissue absorbs the compound; and causing thetissue be in a contracted state and exert a stress on the strip to bendthe strip once the compound is absorbed by the tissue.
 28. The method ofclaim 27, wherein the compound comprises a drug candidate.
 29. Themethod of claim 18, further comprising: adding a compound to the tissueso that the tissue absorbs the compound; and causing the tissue be in arelaxed state once the compound is absorbed by the tissue so that thestrip extends the tissue.
 30. A method of generating a three dimensionaltissue, the method comprising: adding, to a well, a cell suspensionmixture, the well comprising a strip of bendable material, wherein thestrip of bendable material is inserted into the well at a first end andat a second end opposite the first end so that the strip is curved;generating, from the cell suspension mixture, a tissue that is affixedto the first end of the strip and the second end of the strip; andremoving the strip from the well, wherein the strip is configured toexert a stress on the tissue after the strip is removed from the well,and wherein the strip exerts a reduced stress on the tissue before thestrip is removed from the well relative to an increased stress on thetissue after the strip is removed from the well.
 31. A system forintegrated mechanical loading of tissue, comprising: a three dimensionaltissue comprising organic material; and a strip of bendable material,the strip comprising: a first region proximate to a first end of thestrip coupled to the tissue; a second region near a second end of thestrip for coupled to the tissue, the second end being opposite the firstend, wherein the tissue exerts a force on the strip to bend the strip,the force caused by contraction of the tissue, and wherein the stripexerts a stress on the tissue.
 32. The system of claim 31, wherein thetissue is isometrically unconstrained.
 33. The system of claim 31,wherein the tissue is configured to contract by at least 10% an initiallength of the tissue.
 34. The system of claim 31, wherein the tissue isconfigured to contract by at least 20% an initial length of the tissue.35. A method for drug screening on a tissue, comprising: selecting oneor more parameters of a strip to tune a loading value of the strip thatthe strip is configured to exert; generating a tissue that is integratedwith the strip that provides the loading value on the tissue; contactinga compound with the tissue; and measuring an effect of the compound onthe tissue.
 36. The method of claim 35, wherein the compound is amuscarinic agonist.
 37. The method of claim 35, wherein the compound isa stimulant.
 38. The method of claim 35, wherein measuring the effect ofthe compound on the tissue comprises measuring a contractility of thetissue.
 39. The method of claim 35, wherein measuring the effect of thecompound on the tissue comprises measuring a twitch response of thetissue.